EP1526380A1 - Méthode de diagnostic in vitro et de pronostic in vitro du cancer du pancréas et méthode pour le développement de médicaments contre le cancer du pancréas - Google Patents

Méthode de diagnostic in vitro et de pronostic in vitro du cancer du pancréas et méthode pour le développement de médicaments contre le cancer du pancréas Download PDF

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EP1526380A1
EP1526380A1 EP03380236A EP03380236A EP1526380A1 EP 1526380 A1 EP1526380 A1 EP 1526380A1 EP 03380236 A EP03380236 A EP 03380236A EP 03380236 A EP03380236 A EP 03380236A EP 1526380 A1 EP1526380 A1 EP 1526380A1
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protein
efnb2
ednra
pancreas
gene
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English (en)
Inventor
Jokin Del Amon Iribarren
Corina Junquera Sanchez-Vallejo
Jorge Ochoa Garay
Pedro Escuredo Garcia De Galdeano
Simon Santa Cruz
Antonio Martinez Martinez
Laureano Simon Buela
Maria Eladia Arguelles Sanchez
Juan Ramon De Los Toyos Gonzalez
Luis Barneo Serra
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Progenika Biopharma SA
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Progenika Biopharma SA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention refers to an in vitro method to detect the presence of a cancer of the pancreas in an individual, to determine the stage or severity of this cancer in the individual, or to monitor the effect of therapy administered to an individual with the said cancer; to screen for, identify, develop and evaluate the efficacy of therapeutic compounds for this cancer in an attempt to develop new medicinal products and to agents that inhibit expression of the Ephrin-B2 protein and/or the EDNRA protein, and/or the effects of this expression.
  • pancreas Cancer of the pancreas was the cause of more than 220,000 deaths world-wide and more than 3,600 in Spain during the year 2,000 (GLOBOCAN).
  • the clinical behaviour of pancreatic cancer is homogeneous and always unfavourable and there are no clear differences in survival in the different stages. Only very few patients with pancreatic cancer experience favourable outcomes. A possible explanation for this is that even in patients with small tumours, classified as stage I, the disease has spread beyond the pancreas. Its diagnosis in the initial stages, except for exceptional cases, is very difficult. Of patients diagnosed, 75% present the disease in advanced stages (Stages III and IV).
  • Erythropoietin-producing hepatocellular markers are a family of tyrosine-kinase receptors that are activated on interaction with their ligands, the ephrins. Both the ephrins and their ligands are involved in basic processes of nervous system development (Tessier-Lavigne M., 1995, Cell, 82:345-348; Winslow J.W., et al., 1995, Neuron, 14:973-981; Lumsden A., and Krumlauf R., 1996, Science, 274:1109-1115; Magal E., et al., 1996; J. Neurosci.
  • Ephrin-B2 (EFNB2) is a transmembrane protein specifically expressed in arteries that reacts with the EPH B4 receptor, which is expressed more abundantly in veins, resulting in activation of the proliferation and migration stages of angiogenesis (Steinle J.J., et at., 2002, J. Biol.. Chem., 277:43830-43835; gerety S.S., and Anderson D.J., 2002, Development, 129:1397-1410). It has been previously shown that the gene ephrin-b2 (ephrin-b2; GeneBank code U16797.
  • efnb2 which encodes the protein ephrin-B2
  • gastric cancer Kataoka H. et al., 2002, J. Cancer res. Clin. Oncol., 128:343-348
  • neuroblastoma Tang, X.X., et at., 2000, Proc. Natl. Acad. Sci., 97:19936-10941
  • primary and metastatic melanomas Vogt T., et al., 1998, Clin. Cancer Res., 4:791-797)
  • small cell lung cancer Tang, X.X., et al., 1999, Clin.
  • the EFNB2 gene which encodes the protein EFNB2, or the protein EFNB2, is overexpressed in tumoral tissue from individuals with ductal adenocarcinoma of the pancreas.
  • Endothelins and their receptors are proteins with a strong vasoconstrictor activity on mature blood vessels. These proteins have also been shown to be involved in stimulating proliferation and migration of endothelial cells, myocytes and fibroblasts. These proteins have been associated with angiogenesis and their expression has been demonstrated in endothelial cells of tumours of ovary, colon, prostate, cervix, lung, uterus, Kaposi sarcoma, gliomas, meningiomas (Rosano L., et al., 2003, Cancer Res. 63:2447-53; Asham E., 2001, Br. J. Cancer., 85:1759-63; Peduto E. L., 2003, Br. J.
  • endothelins or their receptors or some of these proteins, are overexpressed in tumoral tissue samples from individuals with pancreatic ductal adenocarcinomas.
  • the authors of the present invention have discovered, after thorough research and using different techniques (DNA-chips and quantitative RT-PCR to measure levels of gene expression and western-blotting to measure levels of protein expression), that expression of the efnb2 gene and the expression of the ednra gene, which encodes the endothelin receptor A (EDNRA), are elevated in the cancers of the pancreas when compared with non-tumoral expression in the same individuals or individuals with pancreatitis.
  • the authors of the present invention have also discovered that the concentration of the EFNB2 protein is very high in pancreatic ductal adenocarcinomas, compared with non-tumoral expression in the same individuals, healthy individuals or individuals with pancreatitis. This makes EFNB2 and EDNRA useful targets for the development of new in vitro methods for diagnosis and prognosis and for the identification and development of therapeutic compounds for cancer of the pancreas, especially for pancreatic ductal adenocarcinomas.
  • the present invention therefore, provides a highly sensitive in vitro method to detect the presence of cancer of the pancreas, especially a pancreatic ductal adenocarcinoma, to determine the stage or severity of this carcinoma in an individual or to monitor the effect of therapy administered to an individual with the said carcinoma, based on the detection and/or quantification of the EFNB2 protein, of the efnb2 mRNA or of the corresponding efnb2 cDNA, of the EDNRA protein, of the ednra mRNA, or of the corresponding ednra cDNA, or of any combination of these in a sample of an individual.
  • the present invention provides targets or tools for the screening, identification, development and evaluation of the efficacy of therapeutic compounds for the treatment of cancer of pancreas, especially for ductal adenocarcinoma of the pancreas.
  • the invention provides agents characterised by the fact that they inhibit expression and/or activity of the EFNB2 protein and/or the EDNRA protein for the treatment of cancer of pancreas, especially for pancreatic ductal adenocarcinomas.
  • the object of the present invention is to develop an in vitro method to detect the presence of cancer of the pancreas, especially of pancreatic ductal adenocarcinomas, to determine the stage or severity of this cancer in the individual or to monitor the effect of the therapy administered to an individual with this cancer.
  • a second object of the present invention is an in vitro method to screen for, identify, develop and evaluate the efficacy of compounds to treat cancer of the pancreas, especially pancreatic ductal adenocarcinomas.
  • An additional object of the invention lies in the use of sequences derived from the efnb2 gene to establish the diagnosis and prognosis in vitro of cancer of the pancreas, especially pancreatic ductal adenocarcinoma, and to screen for, identify, develop and evaluate the efficacy of compounds for the treatment of this cancer.
  • Another object of the present invention is to provide agents characterised because they inhibit expression and/or activity of the protein EFNB2, and/or of the EDNRA protein, to treat cancer of the pancreas, especially pancreatic ductal adenocarcinoma.
  • Another object of this invention is a pharmaceutical composition consisting of one or several therapeutic agents together with an acceptable pharmaceutical excipient to treat cancer of the pancreas, especially pancreatic ductal adenocarcinoma.
  • the present invention is based on the discovery that both gene expression of efnb2 and ednra, and the concentration of the EFNB2 and EDNRA proteins are increased in ductal adenocarcinoma of the pancreas of an individual.
  • the present invention first of all provides an in vitro method to detect the presence of a cancer of the pancreas, especially of a ductal adenocarcinoma of the pancreas in an individual, to determine the stage or severity of this cancer in the individual, or to monitor the effect of the therapy administered to an individual with this cancer, that comprises:
  • the method provided by the present invention is highly sensitive and specific and is based on the fact that subjects or individuals diagnosed with cancer of the pancreas, especially with ductal adenocarcinoma of the pancreas, present high levels of mRNA transcribed from the efnb2 gene ( elevated levels of expression of the efnb2 gene ) or elevated levels of the protein coded by the efnb2 gene ( Protein EFNB2 ), or elevated levels of mRNA transcribed from the ednra gene ( elevated levels of expression of the ednra gene ), or elevated levels of the protein coded by the ednra gene ( EDNRA protein), in comparison with the corresponding levels in samples from subjects without a clinical history of this cancer.
  • the present method comprises a step in which a sample is obtained from the individual.
  • Different liquid samples can be used such as: urine, blood, plasma, serum, pleural fluid, ascitic fluid, synovial fluid, bile, semen, gastric exudate or cerebrospinal fluid.
  • the sample can also consist of tissue of the affected organ, such as the pancreas that can be obtained by any conventional method, preferably surgical resection. Samples can be obtained from subjects previously diagnosed or not diagnosed with a specific type of cancer; or also from a subject receiving treatment, or who has previously received treatment for a cancer, especially for ductal adenocarcinoma of the pancreas.
  • the present method also comprises a step for extraction of the sample, either to obtain the extract of proteins from it or to obtain the extract of total RNA.
  • a step for extraction of the sample either to obtain the extract of proteins from it or to obtain the extract of total RNA.
  • One of these two extracts provides the working material for the next phase.
  • the extraction protocols for total protein or total RNA are well known by experts in the area (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532).
  • Any conventional assay can be used in the context of the invention to detect a cancer, provided that it measures in vitro the levels of mRNA transcribed from the efnb2 gene or its complementary cDNA, the concentration of the Protein EFNB2, or the levels of mRNA transcribed from the ednra gene or its complementary cDNA, or the concentration of EDNRA protein, in samples collected from individuals to be studied and control individuals.
  • this invention provides a method to detect the presence of a cancer of the pancreas, especially the presence of ductal adenocarcinoma of the pancreas in an individual, to determine the stage or severity of this cancer in an individual, or to monitor the effect of the therapy administered to an individual who presents this cancer, based either on measuring the levels of the EFNB2 protein or on measuring the level of expression of the EDNRA gene or the level of expression of the ednra gene.
  • the method of the invention comprises a first step in which the protein extract of the sample is placed in contact with a composition of one or more specific antibodies against one or more epitopes of the EFNB2 protein or the EDNRA protein, and a second stage to quantify the complexes formed by antibodies and the EFNB2 protein or the EDNRA protein.
  • immunological assays available to detect and quantify formation of specific antigen-antibody complexes; numerous competitive or non-competitive protein-binding assays have been described previously and a large number of these are available commercially.
  • the EFNB2 protein or the EDNRA protein can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or recombinant fragments of these, combibodies and Fab fragments or scFv of antibodies, specific against the protein EFNB2; these antibodies are human, humanised or of animal origin.
  • the antibodies used in these assays can be labelled or unlabelled; the unlabelled antibodies can be used in agglutination assays; the labelled antibodies can be used in a wide range of assays.
  • Marker molecules that can be used to label antibodies include radionucleotides, enzymes, fluofluorides, chemoluminescent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, colorants and derivatives.
  • assays which use unlabelled antibodies (primary antibody) and labelled antibodies (secondary antibodies); these techniques include the western-blot or western transfer, ELISA (Enzyme-Linked immunosorbent assay), RIA (Radioimmunoassay), Competitive EIA (Competitive enzyme immunoassay), DAS-ELlSA (Double antibody sandwich-ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies or colloidal precipitation in formats such as dipsticks.
  • ELISA Enzyme-Linked immunosorbent assay
  • RIA Radioimmunoassay
  • Competitive EIA Competitive enzyme immunoassay
  • DAS-ELlSA Double antibody sandwich-ELISA
  • immunocytochemical and immunohistochemical techniques techniques based on the use of biochips or protein microarrays that include specific antibodies or colloidal precipitation in formats such as dipsticks.
  • the preferred immunoassay for the invention is a double antibody sandwich ELISA ( DAS-ELISA ). In this immunoassay any antibody or combination of antibodies can be used, specific against one or more epitopes of the EFNB2 protein or the EDNRA protein.
  • a monoclonal or polyclonal antibody, or a fragment of this antibody, or a combination of these antibodies, that cover a solid phase is placed in contact with the sample to be analysed and is incubated for a specific time and in appropriate conditions to form the antigen-antibody complexes. After washing in appropriate conditions to eliminate non-specific complexes, an indicator reagent, consisting in a monoclonal or polyclonal antibody, or a fragment of this antibody, or a combination of these, bound to a signal generator compound, is incubated with the antigen-antibody complexes in appropriate conditions of time and temperature.
  • Presence of the EFNB2 protein or the EDNRA protein in the sample to be analysed is detected and, if present, quantified and the signal generated is measured.
  • the amount of EFNB2 protein or EDNRA protein present in the sample to be analysed is proportional to this signal.
  • the method of the invention effects the detection of the mRNA or of the corresponding cDNA of the efnb2 gene or of the mRNA or the corresponding cDNA of the ednra gene, that comprises a first step of amplification of the RNA that is present in the extract of total RNA, or of the corresponding cDNA synthesised by reverse transcription of the mRNA; and a second step of quantification of the amplification product from either the mRNA or the cDNA of the efnb2 gene, or the mRNA or the cDNA of the ednra gene.
  • mRNA amplification consists in retrotranscribing the mRNA into cDNA (RT), followed by Polymerase Chain Reaction (PCR), using oligonucleotide primers, using the primer sequences 5'-AGCTTGTTTAACGGCAGTGTCAT-3' and 5'- GCAGCAATTTGGCAACCTTT - 3' for efnb2 and 5'- TGCTTGAATTGCAAGGCTAAGAA -3' and 5'-GCGCCAGTGGAATAATAGATTTG -3' for ednra.
  • PCR is a technique for the amplification of a specific nucleotide sequence (target) contained in a mixture of nucleotide sequences.
  • PCR In PCR, an excess of a pair of oligonucleotide primers is used that hybridise with complementary strands of the target nucleotide sequence. After this, an enzyme with polymerase activity (DNA Polymerase) extends each primer, using the target nucleotide sequence as a template. The extension products are, therefore, converted into target sequences, after dissociation of the original strand. New primer molecules hybridise and the polymerase extends them. The cycle is repeated to exponentially increase the number of target sequences.
  • DNA Polymerase enzyme with polymerase activity
  • the amplified product is detected by agarose gel electrophoresis as follows: five microliters of amplification product are separated by agarose gel electrophoresis at a concentration of 2%, in a TBE buffer 0.5x at 100 vdc for one hour. After electrophoresis the gel is stained with ethidium bromide and the amplification product is observed when the gel is illuminated with ultraviolet (uv) light.
  • a preferred method is to transfer the amplified product to a nylon membrane by Southern blotting or Southern transfer techniques to be detected with a specific cDNA probe of the efnb and/or ednra gene, appropriately labelled.
  • mRNA detection is performed following electrophoretic separation of mRNA by transferring the mRNA to a nylon membrane using transfer techniques such as northern-blot or northern transfer and detecting it with specific RNA probes or of the corresponding cDNA of the efnb gene or the mRNA or the corresponding cDNA of the ednra gene.
  • transfer techniques such as northern-blot or northern transfer and detecting it with specific RNA probes or of the corresponding cDNA of the efnb gene or the mRNA or the corresponding cDNA of the ednra gene.
  • amplification and quantification of the mRNA corresponding to the efnb2 gene and/or amplification and quantification of the mRNA corresponding to the ednra gene is carried out by quantitative RT-PCR in real time (Q-PCR).
  • the final step of the method of the invention to detect in vitro the presence of the cancer in a sample from an individual comprises comparing the amount of protein EFNB2, the amount of mRNA of the efnb2 gene or the amount of the corresponding cDNA or the amount of EDNRA protein, the amount of mRNA of the ednra gene, or the amount of corresponding cDNA detected in a sample of an individual, with the amount of protein EFNB2, the amount of mRNA of the efnb2 gene, the amount of corresponding cDNA, with the amount of EDNRA protein, the amount of mRNA of the ednra gene, the amount of corresponding cDNA detected in the samples of control subjects or in previous non-tumorous samples of the same individual or with normal reference values.
  • the invention also provides a method in vitro to identify and evaluate the efficacy of therapeutic agents against a cancer of the pancreas, especially against a ductal adenocarcinoma of the pancreas that comprises:
  • Quantification of the expression levels of the efnb2 gene, the protein EFNB2, the ednra gene or the EDNRA protein is done in a similar way to that described in the method of the invention to detect in vitro the presence of a cancer of the pancreas, especially of a ductal adenocarcinoma of the pancreas in an individual.
  • an agent reduces the expression levels of the efnb2 gene and/or the ednra gene or reverses the effects of high expression of this gene, preferably reducing the levels of cellular proliferation, this agent becomes a candidate for the cancer therapy.
  • another object of this invention refers to the use of nucleotide or peptide sequences derived from the efnb2 and/or ednra gene, in methods to screen for, identify, develop and evaluate the efficacy of therapeutic compounds against cancer of the pancreas, especially ductal adenocarcinoma of the pancreas. It is noteworthy, the recent importance given to screening methods based on the competitive or non-competitive binding of the potential therapeutic molecule to the therapeutic target.
  • Another object of this invention refers to the use of nucleotide or peptide sequences derived from the efnb2 and/or the ednra gene to detect the presence of a cancer of the pancreas, especially of a ductal adenocarcinoma of the pancreas, to determine the stage or severity of these cancers in the individual or to monitor the effect of the therapy administered to an individual with these cancers.
  • Another object of this invention consists in providing agents characterised because they inhibit expression and/or activity of the EFNB2 protein and/or the EDNRA protein. These agents, which can be identified and evaluated according to the present invention, can be selected from the group comprised by:
  • Example 1 Differential analysis of the expression of the efnb2 gene and the ednra gene in samples of pancreas tissue, using Human Genome U133 DNA microarrays
  • GeneChip Test 3 (Affymetrix, Santa Clara) microarrays were used, that permitted the quality of RNA to be tested before analysing expression with the GeneChip Human Genome U133A array (Affymetrix, Santa Clara), which represents 13,220 recorded complete gene sequences;
  • the efnb2 gene is represented in the microarray by the set of probes 202668_at of Affymetrix, which are sense oligonucleotides 25 nucleotides long, designed on the basis of the Hs.30942 sequence of Unigene, or U16797 of GeneBank (Table 1);
  • the ednra gene is represented in the microarray by the set of probes 204464_s_at of Affymetrix, which are sense nucleotides 25 nucleotides long, designed on the basis of the Hs.76252 sequence of Unigene or L06622 of GeneBank (Table 2).
  • Stage III tumour spread to regional lymph nodes and Stage IVB, metastases in distant organs and tissues. All samples were clinically and histologically classified (grade and stage) in the Hospital Central de Asturias, the same hospital where they had been collected according to the precepts of the Helsinki declaration. Samples were frozen in liquid nitrogen immediately after extraction and stored at -80°C until analysis.
  • RNA of each biopsy was obtained by homogenising the tissue in TRlzol® Reagent (Life Technologies), following the supplier's recommendations. The resulting total RNA was cleaned with the Rneasy kit (QIAGEN) (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). Of each preparation of total RNA, 10 ⁇ g were used as starting material for synthesis of the first cDNA strand with the reverse transcriptase enzyme SuperScriptTM II RNase (Life Technologies), using as a primer an oligonucleotide oligo-dT containing the sequence of the RNA polymerase promoter of phage T7.
  • the second cDNA strand was synthesised using the enzymes DNA polymerase I of E . coli (Invitrogen Life Technologies), DNA ligase of E. coli (Invitrogen Life Technologies), Rnase H of E. coli (Invitrogen Life Technologies), and DNA polymerase of phage T4 (Invitrogen Life Technologies).
  • the biotin labelled cRNA was synthesised using the ENZO BioArrayTM HighYieldTM Transcript Labeling Kit (Enzo Diagnostics Inc). After in vitro transcription, the nucleotides not incorporated were eliminated using the RNeasy columns (QIAGEN).
  • a total of 15 ⁇ g of each biotinylated cRNA were fragmented at 94°C for 35 minutes in a buffer solution containing 40 mM Tris-Acetate (pH 8.1), 100mM KOAc and 30mM MgOAc.
  • the fragmented cRNA was mixed with hybridization buffer (100mM MES, 1M NaCl, 20 mM EDTA, 0.01% Tween 20) and heated to 99° for 5 minutes and then to 45° for 5 minutes, after which it was loaded in the Affymetrix array.
  • the first array in which the hybridization was carried out was Test 3 of Affymetrix.
  • RNA was tested before analysing expression in the Affymetrix® GeneChip® Human Genome 133 A (HG-U133A).
  • arrays were incubated in a rotary incubator at 45° for 16 hours with a constant rotation of 60 rpm. Washing and staining of each array was done in the Affymetrix® fluid station. A washing and staining programme was used that included:
  • ii) Change This indicates whether expression of a specific transcript Increases (I), Decreases (D), Does not change (NC), Increases marginally (IM), or decreases marginally (DM), iii) Signal Log Ratio (SLR): This indicates the level of change in expression between the baseline (control) and the sample under test. This change is expressed as the log 2 of the ratio ( fold change or number of times that this gene expression is elevated or repressed in the problem-tumoral sample versus a healthy control sample).
  • SLR value of 1 is considered to be significant (equivalent to a fold change of 2), for transcripts in which expression increases compared to control and of -1, for transcripts for which expression decreases compared to control.
  • Example 2 Differential analysis of expression of the efnb2 gene and the ednra gene in samples of pancreas tissue using Real time - quantitative RT-PCR
  • the method used consists in the reverse transcription of mRNA to cDNA followed by its amplification in a LightCycler (Roche) equipment using SYBR Green to detect the amplified product. Quantification was done in real time and permits the relative expression of the sequence to be calculated in different samples in the linear amplification phase of the reaction.
  • RNA of each of the biopsies was obtained by homogenising the tissue in TRlzol® Reagent (Life Technologies), following the supplier's recommendations. The resulting total RNA was cleaned with the RNeasy kit (QIAGEN) (Chomczynski P. et at., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). The RNA was quantified spectrophotometrically and 5 ⁇ g of total RNA were digested with DNasel.
  • RNA treated with DNAse was used as starter material for the synthesis of the first strand of cDNA with the inverse transcriptase enzyme SuperScriptTM II RNase (Life Technologies), using an oligo-dT oligonucleotide that contained the sequence of the RNA polymerase promoter of the phage T7 as a primer. Aliquots of cDNA diluted to the working concentration were prepared.
  • the cDNA synthesised was amplified using specific primers of the human efnb2 gene (5'-AGCTTGTTTAACGGCAGTGTCAT -3' and 5'-GCAGCAATTTGGCAACCTTT-3'), of the human ednra gene (5'-TGCTTGAATTGCAAGGCTAAGAA-3' and 5'-GCGCCAGTGGAATAATAGATTTG-3') and of the gene that codes for the human ribosomal protein L10 (5'-TGCGATGGCTGCACACA-3' and 5'-TCCCTTAGAGCAACCCATACAAC-3').
  • PCR reactions in real time were prepared using the LightCycler-FastStart DNA master SYBR Green I kit (Roche) following the manufacturer's instructions.
  • the amplification programme consisted in 1 cycle of 95°C (denaturation) for 10 s, 60°C (annealing) for 5 s, 72°C (amplification and acquisition of fluorescence) for 10 s.
  • the programme of analysis of the denaturation curves consists of a cycle of one pulse of 95°C, 65°C for 15 s, and a pulse of 95°C for the amplification and acquisition step.
  • the specificity of the PCR products are determined by analysing the denaturation curves. Subsequently, as a relative measure of gene expression, the ratio of the abundance of mRNA transcripts of efnb2 to the abundance of ribl10 transcripts was calculated and the ratio was normalised for each of the tumoral samples on the basis of the values of the control sample.
  • Ratio E target -(Cp target control -Cp target sample ) E reference -(Cp reference control - Cp reference sample)
  • E the amplification efficiency
  • Cp the Crossing point
  • target is efnb2
  • reference is ribl10
  • control is the control sample (non-tumorous or chronic pancreatitis)
  • sample is the tumoral sample.
  • the same protocol was used to quantify ednra, in comparison with ribl10.
  • Example 3 Differential analysis of expression of the EFNB2 protein in samples of pancreas tissue using the Western blot technique with specific antibodies.
  • Protein extraction Frozen samples were homogenised in RIPA B buffer [sodium phosphate 20 mM (pH 7.4), NaCl 150 mM, Triton X-100 1%, EDTA 5 mM and a combination of protease inhibitors (Roche Diagnostics Inc., Mannheim,Germany)]. Samples were then centrifuged at 15000 x g for 10 minutes at 4°C, to eliminate cellular debris. Then, the supernatant was recovered and the concentration of proteins was estimated by the Bradford assay (Bio-Rad, Hercules, CA, US).
  • EFNB2 was overexpressed in 2 of the 3 ductal adenocarcinomas of the pancreas that were analyzed.
  • the major immunoreactive band was situated at 120 kDa, which corresponded to the mature form of EFNB2 (Tabla 17, Figure 6).

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EP03380236A 2003-10-21 2003-10-21 Méthode de diagnostic in vitro et de pronostic in vitro du cancer du pancréas et méthode pour le développement de médicaments contre le cancer du pancréas Withdrawn EP1526380A1 (fr)

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